S1. miR-200c-deficiency induced biological effects in mammary epithelial cells. S2. Expression of EMT inducers and stemness genes in miR-200c KO or overexpressing cells. S3. miR-200c-deficiency induces Warburg effect in mammary epithelial cells. S4. The mitochondrial oxidative phosphorylation is elevated by miR-200c in basal-like breast cancer cells. S5. Cell growth of miR-200c KO cells is not affected by low-dosage 2DG pretreatment for two days. S6. 2-DG and NADH treatment enhance oxidative phosphorylation in miR-200c deficient MCF12A cells. S7. NADH treatment fails to affect EMT-phenotype in miR-200c-deficient MCF12A cells. S8. p53 hot-spot mutations induce Warburg effect in mammary epithelial cells. S9. Examples of metabolites affected by miR-200c KO or MTp53-R280K overexpression. S10. miR-200c deficiency leads to altered expression of metabolic genes in central metabolic pathway. S11. PCK2 expression is regulated by ZEB1 and BMI1 in miR-200c-KO or p53 mutated mammary epithelial cells and basal-like breast cancer cells. S12. Expressional correlation between PCK2, miR-200c, and miR-200c direct target ZEB1/ ZEB2 in breast cancer patients. S13. PCK2 expression shows a slight to moderate negative association with EMT and stemness markers in breast cancer patients. S14. Interference of PCK2 expression compromised miR-200c-exeted tumor suppressive effect on p53 mutated BLBC cells.
ARTICLE ABSTRACT
miR-200c is a tumor suppressor miRNA that plays a critical role in regulating epithelial phenotype and cancer stemness. p53 deficiency downregulates the expression of miR-200c and leads to epithelial-mesenchymal transition (EMT) and stemness phenotype, which contributes to the progression of breast cancers. In this study, we demonstrated that CRISPR-mediated knockout (KO) of miR-200c induces metabolic features similar to the metabolic rewiring caused by p53 hot-spot mutations, and that impairing this metabolic reprogramming interferes with miR-200c deficiency–induced stemness and transformation. Moreover, restoring miR-200c expression compromised EMT, stem-cell properties, and the Warburg effect caused by p53 mutations, suggesting that mutant p53 (MTp53) induces EMT-associated phenotypes and metabolic reprogramming by downregulating miR-200c. Mechanistically, decreased expression of PCK2 was observed in miR-200c– and p53-deficient mammary epithelial cells, and forced expression of miR-200c restored PCK2 in p53 mutant–expressing cells. Reduced PCK2 expression not only led to attenuated oxidative phosphorylation (OXPHOS) and increased stemness in normal mammary epithelial cells but also compromised the enhanced OXPHOS and suppression of cancer stemness exerted by miR-200c in p53 mutation–bearing basal-like breast cancer (BLBC) cells. Clinically, PCK2 expression is negatively associated with EMT markers and is downregulated in basal-like subtype and cases with low miR-200c expression or p53 mutation. Notably, low expression of PCK2 is associated with poor overall survival (OS) in patients with breast cancer.
Together, our results suggest that p53 and miR-200c regulate OXPHOS and stem/cancer stemness through PCK2, and loss of the p53–miR-200c–PCK2 axis might provide metabolic advantages that facilitate cancer stemness, leading to the progression of BLBCs.
